盐酸胍和尿素诱导溶菌酶变性的微量热研究

应用恒温微量热技术,对盐酸胍和尿素与溶菌酶在30℃水溶液中的结合作用及造成溶菌酶变性的过程进行了研究,并根据简单结合模型,计算了它们之间的结合常数、结合自由能。用变性中点的直线外推方法求出了表观变性焓。实验结果表明,盐酸胍和尿素诱导溶菌酶变性的相互作用均分为3个阶段。溶菌酶在盐酸胍溶液中的变性焓在pH=4.16时为81...     

Denaturing Effects of Urea and Guanidine Hydrochloride on Hyperthermophilic Esterase from Aeropyrum pernix K1

Introduction Esterases(EC3.1.1.x)representadiversegroup ofhydrolasescatalyzingthecleavageandformationof esterbonds.Theyarewidelydistributedinanimals,plantsandmicroorganisms.Becauseoftheiractivities inbothaqueousandnonaqueoussolventsystems,ester aseshavebe…     

脲和盐酸胍诱导的牛碳酸酐酶B的去折叠

采用非变性聚丙烯酰胺凝胶电泳、十二烷基硫酸钠-聚丙烯酰胺凝胶电泳、高效凝胶排阻色谱和激光光散射光谱研究了脲和盐酸胍诱导的牛碳酸酐酶B的去折叠.实验结果表明,在脲和盐酸胍诱导的牛碳酸酐酶B的去折叠过程中,溶液中只含有单分子牛碳酸酐酶B和二分子牛碳酸酐酶B集聚体;二分子牛碳酸酐酶B集聚体是通过单分子牛碳酸酐酶B之间的疏水和...     

脲和盐酸胍诱导的卵清溶菌酶分子去折叠过程中各稳定构象态的分布和过渡

以内源荧光光谱和荧光相图法研究了脲和盐酸胍诱导的卵清溶菌酶分子的去折叠过程,结果表明,当变性液中脲和盐酸胍的浓度分别约为4.0和3.0 mol/L时,卵清溶菌酶分子的去折叠过程均存在一个折叠中间态,这两个去折叠过程均符合"三态模型".在卵清溶菌酶分子"三态"去折叠过程的基础上,通过变性剂分子和卵清溶菌酶分子之间的缔合一...     

Unfolding and Inactivation of Abalone (<Emphasis Type="Italic">Haliotis diversicolor</Emphasis>) Alkaline Phosphatase During Denaturation by Guanidine Hydrochloride

Abalone, a kind of low poikilothermic invertebrate, is easily exposed to ocean environment stress. Since it is one of the important mariculture animals, the attention paid to the abalone study becomes increasing. Alkaline phosphatase (ALPase, EC 3.1.3.1) is a kind of zinc-contained metalloenzyme, which catalyzes the nonspecific hydrolysis of phosphate monoesters. Unfolding and inactivation of ALPase from abalone (Haliotis diversicolor) during denaturation by guanidine hydrochloride (GuHCl) of different concentrations has first been studied. The kinetic theory of the substrate reaction by enzyme was described by Tsou, which was applied to the study on ALPase’s kinetic course of inactivation by GuHCl. The result showed that the inactivation of the enzyme by GuHCl was a slow, reversible reaction with fractional remaining activity. The microscopic rate constants were determined. The result, , showed that the enzyme was protected by the substrate to a certain extent during guanidine denaturation. The changes of conformation of the enzyme in different concentrations of GuHCl have been studied by means of measuring the fluorescence spectra. The results showed that the inactivation occurred before the noticeable conformational changes of the enzyme molecule as a whole can be detected, which suggests that the active site of the enzyme has more flexibility than the whole enzyme molecule. These studies will facilitate the understanding of physiological and biochemical features of the H. diversicolor and will also help in the understanding of the abalone immune system.     

盐酸胍对反胶束中RNase A活性的影响

以胞嘧啶核苷酸2',3'-环单磷酸酯为底物研究了变性剂盐酸胍对十二胺丁酸盐-环己烷反胶束溶液中核糖核酸酶A活性的影响,同水溶液相比,盐酸胍对反胶束中酶活性的抑制作用很小。反胶束的大小限制了酶分子天然态构象的改变,从而保证了其活性中心的完整性。核糖核酸酶A的内源荧光研究发现,同水溶液相比,反胶束中蛋白的最大发射波长没有发生变化,但荧光偏振极化度增加,也表明了在反胶束中酶分子的运动自由度较在水溶液中有所降低。     

盐酸胍对疏水色谱中蛋白质保留行为的影响

研究了变性剂酸胍对几种蛋白质在高效疏水色谱中保留行为的影响，用液相色谱中溶质的计量置换保留模型和测流动相表面张力及蛋白质紫外吸收光谱方法研究的结果表明：在低浓度范围内，盐酸胍主要以疏水作用影响蛋白质的保留；而在高浓度区域内，盐酸胍的存在则显著影响蛋白质分子的构象，使其保留行为发生变化。     

Ultrasensitive Fluorescence Determination of Adenosine Deaminase using DNA-Templated Silver Nanoclusters and Isothermal Amplification

Highly sensitive determination of proteins is essential to biomedical research and clinical diagnosis. Here a new fluorescence strategy for protein assays is reported that utilizes the exponential amplification reaction and silver nanocluster label-free architecture. Superior sensitivity was obtained for adenosine deaminase with a detection limit of 0.8 milliunit per liter. This method was employed for the determination of adenosine deaminase in human serum. The method displayed high selectivity for adenosine deaminase compared to other proteins, thus providing a promising alternative to standard approaches for the determination of proteins.     

脲和盐酸胍诱导过氧化氢酶去折叠的研究

用荧光相图法分别研究了脲和盐酸胍诱导牛肝过氧化氢酶去折叠的过程。当脲 浓度从0依次增大至0.50,4.5和8.0 mol/L时,过氧化氢酶从天然四聚体依次转变 为蓬松的四聚体、部分折叠的无活性二聚体和去折叠态,而当盐酸胍浓度从0依次 变化至0.65,2.5和6.0 mol/L时,过氧化氢酶则从天然四聚体集资转变为部分折叠 的激活二聚体、部分折叠的单体和去折叠态,这表明无论是用脲还是用盐酸胍作为 变性剂,该蛋白的变性过程都符合“四态模型”,但这两种变性剂诱导该蛋白去折 叠的途径和机制有较大差异。实验结果表明荧光相图法可以检测蛋白质去折叠的中 间态。用等温滴定量去热法研究了盐酸胍诱导过氧化氢酶去折叠过程的热力学, 25.0 ℃时低浓度盐酸胍诱导该蛋白从天然四聚体转变为部分折叠的激活二聚体的 本征摩尔构象变化焓、Gibbs自由能和熵分别为-69.2 kJ·mol~(-1),6.43 kJ· mol~(-1)和-254 J·K~(-1)·mol~(-1),据此推断盐酸胍通过熵效应和静电效应来 稳定和激活该二聚体。     

盐酸胍诱导的变性卵清溶菌酶分子重折叠过程及其各稳定构象态的分布和过渡

采用变性和非变性电泳、 高效凝胶排阻色谱、 内源荧光发射光谱和荧光相图以及生物活性测定等方法, 研究了盐酸胍诱导的变性卵清溶菌酶分子的重折叠过程及此过程中卵清溶菌酶分子各稳定构象态的分布和过渡. 结果表明, 当复性液中盐酸胍浓度分别约为5.0和2.4 mol/L时, 变性卵清溶菌酶分子的重折叠过程各存在1个稳定折叠中间态, 重折叠过程符合"四态模型". 在卵清溶菌酶分子四态重折叠过程基础上, 结合盐酸胍与卵清溶菌酶分子之间的缔合-解离平衡, 给出了一个定量描述变性剂诱导的蛋白质分子复性过程中蛋白质分子复性率随溶液中变性剂浓度变化的方程. 该方程包含2个特征折叠参数, 一个是蛋白质分子从一个稳定构象态过渡到另一个稳定构象态的热力学过渡平衡常数k; 另一个是在此过程中平均每个蛋白质分子所结合的变性剂分子数目m. 通过这2个特征折叠参数能够定量描述盐酸胍诱导的变性卵清溶菌酶完全去折叠态、 折叠中间态和天然态分子随复性液中盐酸胍浓度变化的分布和过渡情况.     

Guanidine hydrochloride:An active and simple catalyst for Mannich type reaction in solvent-free condition

Commercially available guanidine hydrochloride(GuHCl) has been found to be a highly efficient catalyst for the Mannich reaction.β-Amino carbonyl compounds were obtained in reasonable yields when the Mannich reaction was carried out at room temperature under solvent-free conditions.     

Comparison Between Catalytic Activity and Activity Changes During Denaturation by Guanidine Hydrochloride of Enzymes in Crystalline State and in Solution

The catalytic activity and activity changes during denaturation by guanidine hydrochloride of glyceraldehyde-3-phosphate dehydrogenase,lactate dehydrogenase and α-chymotrypsin in crystalline state and in solution have been compared.The catalytic activities are lower in crystalline state than in solution. Enzymes in crystalline state are more stable than in solution during denaturation by guanidine hydrochloride.Ammonium sulfate has different effects on catalytic activities of different enzymes and shows protection on all enzymes studied during denaturation by guanidine hydrochloride.The protection is more obvious at high concentrations of guanidine hydrochloride than at low concentrations.It is suggested that the flexibility or mobility of enzyme is required for the catalytic activity and related to the stability of enzymes. Enzymes with less flexibility or mobility are more stable.     

脲和盐酸胍诱导溶菌酶去折叠的荧光相图法研究

用荧光相图法分别研究了脲和盐酸胍诱导卵清溶菌酶去抓叠的过程。当变性体 系中无还原剂2－巯基乙醇存在、脲浓度从0变化至4.0 mol/L（或盐酸胍浓度从0变 化至3.0 mol/L）时,溶菌酶从天然态转变为部分折叠中间态,当脲浓度从4.0 mol/L变化至8.0 mol/L（或盐酸胍浓度从3.0 mol/L变化至6.0 mol/L）时,溶菌 酶从中间态转变为去折叠态,此时该蛋白的变性过程符合“三态模型”。而当变性 体系中有该还原剂存在时,溶菌酶则由天然态直接转变为去折叠态,此时脲诱导该 蛋白去折叠的过程符合曲型的“二态模型”。实难结果表明荧光相图法可以检测蛋 白南去抓叠的中间态。     

腺苷脱氨酶活性分析方法研究进展

腺苷脱氨酶(ADA)是一种核酸代谢酶,与机体细胞免疫活性有重要关系,在很多疾病中,ADA活性均表现异常。因此,越来越多的科研工作者聚焦于ADA活性与疾病关系的研究,并一致认为ADA活性分析对临床疾病的诊断与治疗具有非常重要的意义。该文综述了比色法、色谱法、适配体传感器等ADA活性分析方法的研究进展,以期为ADA的活性分析提供参考。     

Unfolding of Bovine Heart Cytochrome c Induced by Urea and Guanidine Hydrochloride

The unfolding of bovine heart cytochrome c induced by urea and guanidine hydrochloride was studied through their intrinsic fluorescence emission spectra, fluorescence phase diagrams, fluorescence quenching, size‐exclusion chromatographies, native polyacrylamide gel electrophoreses and deactivation profiles. The results showed that during their unfolding in urea and guanidine hydrochloride solutions, bovine heart cytochrome c molecules existed only in a unimolecular form and their bi‐molecular and/or poly‐molecular aggregates and aggregate precipitates were not formed all along. When the urea and guanidine hydrochloride concentrations in denaturation solution were separately about 6.0 and 3.0 mol/L, they could be completely deactivated and almost all of the tryptophan residues originally embedded in the interior of their molecules were exposed to the surface of their molecules. Different from the unfolding of the most often used horse heart cytochrome c, that of bovine heart cytochrome c induced by urea and guanidine hydrochloride was separately a completely co‐operative procedure and followed a two‐state model.     

Ornithine Carbamoyltransferase Unfolding States in the Presence of Urea and Guanidine Hydrochloride

Ornithine carbamoyltransferase folding/unfolding is a complex and not completely understood process. Our experimental results suggest that ornithine carbamoyltransferase interacts in a completely different way with urea and guanidine hydrochloride. In fact, we noticed that, increasing concentration from 0.0 to 8.0 M of the two additives, the enzyme follows a simple one-step transition mechanism in the presence of guanidine hydrochloride, with two macroscopic states (the native and the denatured one) significantly populated, whereas in the presence of urea a lot of different protein states can be detected and analyzed. Circular dichroism and UV-visible spectroscopy reveal a similar mechanism of perturbation at high temperature, with opening of hydrophobic core and a significant loss in α-helix structure in the presence of guanidine hydrochloride that cannot be found in the presence of urea.     

The Use of Chloroformamidine Hydrochloride as a Reagent for the Synthesis of Guanidines from Electron Deficient Aromatic Amines

In efforts to optimize a manufacturing process for an internal development compound, a clean, efficient approach to guanidine synthesis using chloroformamidine hydrochloride was identified. To investigate the general utility of this methodology towards electron‐deficient aromatic amines, a set of favorable conditions were developed from a series of screens, and the scope of the reaction was probed. The successful application of this chemistry to a variety of pyridines, anilines, and heterocyclic compounds highlights its use as an improved, alternative guanylation method for this often challenging set of aromatic amines.     

Spectral Studies on the Conformational Transitions of Bovine Insulin During Denaturant-induced Unfolding

Transitions among various molecule states and conformational changes of bovine insulin were investigated under different denaturing conditions by means of fluorescence phase diagrams,fluorescence quenching,1-anilinonaphthalene-8-sulfonate（ANS） binding assay and circular dichroism（CD） spectra.In both guanidine hydrochloride（GuHCl）-and urea-denatured procedures,the spatial structure of insulin molecules changed from ordered states to relative unordered ones with the increasing of denaturant concentration.The GuHCl-denatured process followed a four-state model,for there were two intermediates existed in 2.0 and 6.0 mol/L GuHC1,respectively.Intermediate I1 is more compact than the normal protein.And intermediate I2 has lost most of the secondary structures.When GuHCl concentration was above 6.0 mol/L,the fluorophores originally existed in the internal of insulin molecules would expose to the surface.However,the urea-denatured process followed a three-state model,only one intermediate existed in 2.5 mol/L urea.During the urea-denatured procedure,the fluorophores originally existed in theinternal of insulin molecules didn＇t expose to the surface.     

牛血清蛋白在盐酸胍和尿素体系中变性的微量热研究

在30 ℃时用恒温微量热法研究了不同pH值下盐酸胍、尿素诱导牛血清蛋白变性的过程. 并用Privalov提出的简单键合模型对量热数据进行了分析, 计算了表观键合常数K, 简单键合的单个表观键合自由能ΔG和总吉布斯能ΔG(a), 用变性中点的直线外推方法求出了表观变性焓ΔH_d. 实验结果表明, 牛血清蛋白与盐酸胍的键合在碱性条件下更易进行, 牛血清蛋白在盐酸胍溶液中的变性焓ΔH_d在牛血清蛋白的pH＝6.97和7.05时为350 kJ•mol^－1, 在pH＝9.30时为275 kJ•mol^－1, 表明牛血清蛋白在接近中性时较稳定. 而牛血清蛋白与尿素的键合在酸性条件下更易进行, 此变性焓ΔH_d在牛血清蛋白的pH＝6.97时为295 kJ•mol^－1, 在pH＝7.05和9.30时为230 kJ•mol^－1. 此结果说明牛血清蛋白在两种变性剂溶液中的展开程度是不同的.     

脲或盐酸胍溶液中变性溶菌酶复性过程中集聚体的研究

建立在蛋白质变性-复性三态模型的基础上, 给出了一个描述在变性液中变性蛋白质复性时蛋白质浓度和其复性率的关系式. 通过这个关系式, 可以获得两个重要的描述蛋白质变性-复性体系特征的参数, 一个是包含在一个集聚体分子中的变性蛋白质的分子数目n, 另一个是蛋白质从原始态到形成集聚体过程中的表观集聚平衡常数K. 以三种溶菌酶在脲和盐酸胍溶液中的变性-复性过程对此方程进行了验证, 结果表明所给出的方程能够很好地描述三种溶菌酶在这两种变性液中的复性结果, 三种溶菌酶在两种变性液中有形成二分子集聚体的趋势. 变性溶菌酶在复性过程中的电泳和高效凝胶排阻色谱也同时能够监测到复性过程中集聚体的形成, 并且监测结果与上述方程所得的结果一致.